The unexpected encounter, in 1995, between the fields of HIV and chemokines has dramatically advanced our understanding of AIDS pathogenesis, opening new perspectives for the development of effective prophylactic and therapeutic measures against HIV. The classic HIV-suppressive chemokines were shown to act through the recognition and blockade of specific viral coreceptors expressed on the surface of susceptible cells. Thus, RANTES, MIP-1alpha; and MIP-1beta, selectively inhibit HIV isolates that use their respective receptor, CCR5, as a coreceptor, while SDF-1 selectively inhibits those using its specific receptor, CXCR4. Owing to their inherent antiviral properties, analogues or functional mimics of these chemokines may be exploited for the development of novel anti-HIV therapeutics or preventive topical microbicides. 1) Identification of two novel anti-HIV chemokines with a broad spectrum of action: CXCL4/platelet factor-4 and XCL1/lymphotactin. Using large-scale and multi-array screening methods, we recently identified two novel HIV-suppressive chemokines, CXCL4/platelet factor-4 and XCL1/lymphotactin. CXCL4 is produced by platelets and is the most abundant protein contained within the platelet alpha-granules, while XCL1 is primarily produced by CD8+ T cells upon stimulation. XCL1 is a member of the C-chemokine family and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that treatment with either human CXCL4 or XCL1 causes a dose-dependent inhibition of HIV-1 infection in different experimental systems including primary CD4+ T cells, macrophages, and continuous CD4+ cell lines such as MAGI and TZMbl. Of note, these two novel antiviral chemokines are both active on a broad spectrum of HIV-1 isolates, with similar potency against viruses that use CCR5 and those that use CXCR4 as coreceptors. These results identify CXCL4 as a novel HIV-suppressive chemokine with an atypically broad spectrum of antiviral activity, opening new perspectives for therapy and prevention. 2) Elucidation of the mechanism of antiviral action of CXCL4 and XCL1. Our initial approach toward elucidating the mechanism of anti-HIV action of CXCL4 and XCL1 was to identify which step in the viral replication cycle is inhibited by these chemokines. Kinetic experiments using the MAGI assay demonstrated that CXCL4 blocks an early step in the viral replication cycle. Thus, we used an HIV-1 entry assay to demonstrate that CXCL4 is a potent inhibitor not only of viral entry but also of virion attachment to the surface of susceptible cells. Of note, inhibition of entry of different HIV-1 isolates correlated with their sensitivity to CXCL4-mediated inhibition in infection assays. Similar results were obtained with XCL1, which potently inhibited both attachment and entry of different biological variants of HIV-1. Using a virion-capture assay with the chemokines immobilized on the surface of immunomagnetic beads, we found that both CXCL4 and XCL1 can specifically bind to intact HIV-1 virions irrespective of their coreceptor-usage phenotype. Further tests demonstrated that CXCL4 and XCL1 are both able to co-immunoprecipitate the gp120 envelope glycoprotein, thus providing evidence for a direct interaction between these chemokines and the gp120 envelope glycoprotein if HIV-1. Furthermore, by virion-capture competition using a panel of gp120-specific mAbs, we were able to map the CXCL4-binding site on gp120 to a region that is in close proximity to, but not overlapping with the CD4-binding domain. In contrast, extensive mapping attempts using multiple anti-gp120 antibodies failed to provide information about the putative binding site of XCL1 on gp120. Altogether, these results demonstrated that CXCL4 and XCL1 inhibit the attachment and entry steps of the HIV-1 life cycle through a novel mechanism that involves direct binding to the viral envelope rather than to the cellular coreceptors. 3) Structure-function analysis of XCL1. To investigate the structure-function relationships in XCL1, we used a series of XCL1 mutants generated and characterized in Dr. Volkman's laboratory in Wisconsin. Experiments with structurally-stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-beta conformation of XCL1 (mutant W55D), which interacts with glycosaminoglycans (GAGs) but does not bind/activate the specific XCR1 receptor, while the classic conformation (mutant CC3), which adopts the typical chemokine fold, is inactive. Furthermore, a systematic analysis of individual point-mutations of most solvent-exposed positively charged residues of XCL1 demonstrated that a region that overlaps with, but is not identical to the GAG-interactive region characterized in previous studies by the Volkman group. Since we demonstrated that the antiviral activity of XCL1 depends on the all-beta conformation (W55D), we examined the inhibitory activity of XCL1 in PBMC infected with X4- or R5-tropic HIV-1 following digestion of cell-surface GAGs with heparitinase. We observed that both WT and W55D XCL1 were equally effective at blocking HIV-1 infection in heparitinase-treated and -untreated cells, while in contrast the CC3 variant remained inactive in both conditions. These data provide further evidence for an antiviral mechanism mediated by direct interaction of XCL1 with the viral envelope, irrespective of its binding to GAGs and/or other structures expressed on the target cell surface. 4) Correlation between CXCL4 levels and clinical/immunological parameters of disease progression in HIV-infected subjects. CXCL4 is primarily produced by megakaryocytes and platelets, and is promptly released by platelets upon activation. Although its primary function is to promote blood coagulation, CXCL4 has multiple, seemingly unrelated, activities including blockade of angiogenesis, activation of immune cells and, as we discovered, inhibition of HIV. Of note, a variety of platelet abnormalities have been described in patients with HIV infection, whose severity correlates with the progression of the immunodeficiency. As a first step toward elucidating the clinical relevance of CXCL4 as an endogenous HIV-suppressive factor, we measured the serum levels of CXCL4 in a cohort of HIV-infected subjects (n = 279) selected to represent different clinical stages of HIV infection. Linear regression analysis showed that serum CXCL4 concentrations were positively correlated with peripheral blood CD4+ T-cell counts (p = 0.0008), CD8+ T-cell counts (p <0.0001) and platelet counts (p <0.0001), and negatively correlated with levels of HIV plasma viremia (p = 0.0156) and C-reactive protein (p = 0.0001). In multivariate regression analysis, the ranks of CXCL4 were found to be independently associated with the CD4+ T-cell ranks (p = 0.0016). These data are compatible with a potential in vivo protective effect of CXCL4. However, it has to be emphasized that the serum levels of CXCL4 do not represent actual circulating levels of the chemokine, but rather the chemokine reservoir harbored by circulating platelets, indicating that subjects with less advanced HIV disease are endowed with a larger storage pool of CXCL4. Although we cannot exclude that the true plasmatic levels of CXCL4 may also be elevated in the early stages of HIV-1 infection, the current technology does not permit to obtain reliable measurements without the interference of contaminating platelets. Additional studies will be important to elucidate the role of CXCL4 in the natural history of HIV disease.